摘要
目的:通过异硫氰酸荧光素(fluorescein isothiocyanate, FITC)标记分离得到的鸡白痢沙门菌噬菌体PC79-13,建立荧光噬菌体检测鸡白痢沙门菌的流程。方法:从污水中分离得到一株鸡白痢沙门菌(Salmonella pullorum)的烈性噬菌体,透射电镜表征其形貌,液体培养宿主后感染测定噬菌体一步生长曲线、热稳定性、pH稳定性、紫外线稳定性及最佳感染复数,以及利用生物信息学方法分析其基因组,了解其生物学特性和基因特征。最后用FITC标记此噬菌体获得一种荧光噬菌体探针。结果:此株鸡白痢沙门菌噬菌体(命名为PC79-13)可在鸡白痢沙门菌C79-13双层琼脂平板上形成大小均一、清晰而透亮的噬菌斑。电镜观察显示:噬菌体PC79-13头部为直径约60 nm的正二十面体,长尾,属于有尾噬菌体属长尾噬菌体科。以鸡白痢沙门菌C79-13为宿主菌测得该噬菌体最佳感染复数为0.01,一步生长曲线显示噬菌体PC79-13的潜伏期为30 min,爆发期为80 min。此外,噬菌体PC79-13对酸碱环境皆有较大耐受性,pH=7.0为其最适酸碱度。其抗紫外线照射和高温能力也较强。FITC标记的荧光PC79-13,宿主鸡白痢沙门菌C79-13感染滴度有少量损失(小于10%)。荧光标记的噬菌体PC79-13能高效结合鸡白痢沙门菌C79-13及其他3株鸡白痢沙门菌,在荧光显微镜下显示绿色荧光棒状结构。荧光PC79-13不结合实验室保存的4株鼠伤寒沙门菌、4株肠炎沙门菌及52株大肠杆菌菌株。结论:分离到一株特异性裂解鸡白痢沙门菌的噬菌体PC79-13。荧光标记的PC79-13仍然保持特异性吸附实验室保存的4株鸡白痢沙门菌菌株的能力,在暗场显微镜下可见目标沙门菌呈现亮绿色荧光,检测限为10 CFU/mL,检测时间少于20 min。因此,构建的荧光噬菌体快速检测法,有潜力未来应用于临床常见鸡白痢沙门菌快检。
Objective:To establish a protocol for detecting Salmonella pullorum with fluorescently labeled bacteriophage PC79-13.Methods:A lytic bacteriophage specific for Salmonella pullorum(abbreviated as S.pullorum)was isolated from sewage.Its morphology was characterized by transmission electron microscopy(TEM).The infectivity of the bacteriophage was assessed by generating a one-step growth curve after liquid culture with the host organism.Additionally,its thermal stability,pH stability,UV stability,and optimal infectious dose were measured,and the bioinformatics methods were used to understand its biological characteristics and genetic features.Finally,the bacteriophage was labeled with FITC to generate a fluorescent probe to detect Salmonella pullorum.Results:The isolated bacteriophage PC79-13 formed uniform,clear plaques on double-layer agar plates containing S.pullorum C79-13.Electron microscopy showed that PC79-13 has an icosahedral head about 60 nm in diameter and a long tail,placing it in the Myoviridae family.The optimal multiplicity of infection(MOI)for PC79-13 was 0.01.The one-step growth curve showed a latent period of 30 min and a burst period of 80 min.PC79-13 exhibited significant tolerance to a range of pH levels,with pH 7.0 being the most suitable.It also showed strong resistance to UV radiation and high temperatures.The FITC-labeled PC79-13 retained its infectivity against S.pullorum C79-13.It bound efficiently to S.pullorum strains,showing green fluorescent rod-like structures under the fluorescence microscope,but did not bind to non-host bacteria such as Salmonella typhimurium,Salmonella enteritidis,and Escherichia coli.Conclusion:PC79-13,a highly lytic and specific bacteriophage against Salmonella pullorum,was isolated.Fluorescently labeled PC79-13 can specifically and rapidly attach to the host Salmonella pullorum,facilitating its detection by fluorescence microscopy with a detection limit of 10 CFU/mL and a detection time of 20 min.Therefore,the fluorescent phage rapid detection method constructed
作者
陆思源
曾宇坤
周昕
LU Siyuan;ZENG Yukun;ZHOU Xin(College of Veterinary Medicine,Yangzhou University,Yangzhou 225009,China)
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
2024年第8期105-115,共11页
China Biotechnology
基金
国家自然科学基金(32271435)
扬州大学大学生创新创业训练计划(C202211117012Y)资助项目。
